INTRODUCTION:

Anxiety disorders are one of the most common mental health issues. In the last ten years, it has affected one-eighth of the world's population and has become an important field of study in psychopharmacology^1,24. The classification of diseases defined by extreme or pathological anxiety as the primary factor influencing mood or emotional state^2,23. Fear is an inherent defensive behavior that allows a person to retreat from a potentially dangerous circumstance.

This disrupts daily activities and is defined as anxiety when it occurs for an extended period of time in an improper situation^3,15,16. Anxiety symptoms include: panic attacks, Sweating, worry, uneasiness, and shortness of breath^4,17,18.

Herbal remedies have a significant impact on global health programs. There are over 450 genera and 6500 species in the family Rubiaceae, which includes plants and tree shrubs. Fundamentally the treatment approach of Ayurvedic medicine is prophylactic and preventative. Ayurvedic herbs like Manjistha are described in Sushruta and Charaka. Rubia cordifoliais called Manjistha in Sanskrit. This plant has thin red bark and very long, flexuous, cylindrical roots (Figure 1). Rubia cordifolia located in Asian nations such as India, china and Afghanistan predominantly inhabits mountainous parts of the northwestern Himalayas, extending eastward up to an elevation of 2500 meters. It’s also found in Greece and Africa⁵.

Rubia cordifolia an ayurvedic herb is a perennial climber with very long cylindric flexible roots and thin red bark. Stems are lengthy rough grooved and slightly woody around the base. The bark is white; the branches are scandent, quadrangular, 3.5cm long and grouped in four whorls; oval,sharp bottom leaves are bigger than the higher chemical elements. Flowers are tiny and white or greenish found in terminal panicles or cymes. Fruits are globose, dark purple or black⁶.

Figure 1: Rubia cordifolia (Manjistha)

MATERIALS AND METHODS:

Plant material: Leaves part of Rubia cordifolia was used for study.

Collection of Plant Material:

Rubia cordifolia leaves were collected from Karnataka. After the leaves were obtained the adhering soil particles were separated from the plant portion, and leaves were carefully removed and cleaned. The leaves were shade dried and crushed mechanically, and the resulting powder underwent several types of tests and investigations.

Authentication of Plant:

The leaves of Rubia cordifolia were collected, and the specimen was certified by Mr. Vinay Ranjan, Scientist-E & Head of office, BSI Central Regional Centre, located near 10 Chatham Lines, Prayagraj 212002. A specimen with voucher number SIP/2025/230 has been deposited at the Botanical Survey of India, Prayagraj.

Preparation of Plant Extract:

The collected plant materials were thoroughly washed in tap water. The cleaned, healthy collected plant samples were cut into small pieces and dried under shade for 2 to 3 weeks at room temperature and cut using a mixer grinder. The powder was maintained in a dry place in an airtight container. The Soxhlet method was used. Leaves powder of Rubia cordifolia are placed in thimble within the extraction chamber 500ml distilled water was used to extract 100gm of powdered Rubia cordifolia leaves, which was then left for 24hours with heating mantle at the temperature of 60^◦C. After several cycles the solvent in the flask becomes enriched with the extract compound.

The extract was then placed on a rotary evaporator to separate the solvent and concentrate it. A freezer was used for freeze-drying. During the experiment, an amount of crude medicine was weighted and diluted in distilled water to the appropriate doses.

Phytochemical Screening:

The crude extract was subjected to phytochemical screening using standard procedures to identify the presence of secondary metabolites, such as glycosides, anthraquinones, alkaloids, phenolics, tannins, saponins, flavonoids, and terpenes.

Drugs:

Standard drug (diazepam) Piramal healthcare Ltd. was obtained from Utthan Shambhunath Hospital Pharmacy, used 2mg/kg dose. Meta-Chlorophenyl piperazine used as anxiety inducing agent, 5mg/kg doses. Distilled water chosen as the vehicle and used to prepare the suspension of extract test doses.

Animal used:

Albino Wistar rats (weight 150-200gm) they had unlimited access to water and a pellet meal, and their temperature was kept at a controlled room temp. of 24±2℃ on a 12:12hour light/dark cycle⁷. The experiment was done in between 9:00 am to 4:00 pm. The institutional animal ethics committee authorized the experimental protocol, and the animal were looked up in accordance with CCSEA norms⁴.

Acute Toxicity Study:

Aqueous extracts of Rubia cordifolia leaf were given to Wistar rats at dosage of 5mg/kg, 50mg/kg, 300mg/kg, and 2000mg/kg without causing any adverse reactions or fatalities during the acute toxicity study.

Animal will be divided into 5 groups. Each group contains 4 animals (Table 1).

Table 1: Dosing and Grouping of Animals

Group	Control Type	Drug/Dose
1.	Normal Control	Distilled water 10ml/kg
2.	Disease Control	mCPP 0.5mg/kg+ distilled water
3.	Standard	2mg/kg/day diazepam
4.	Test Control 1	100mg/kg low dose of Rubia cordifolia
5.	Test Control 2	200mg/kg high dose of Rubia cordifolia

Model for anxiety Elevated plus Maze:

E.P.M (Elevated plus maze) is the most widely used behavioural test to investigate fear and anxiety-related reactions in rodents, including mice and rats. With two opposing enclosed arms (50×10cm) and two competing extended arms (50×10×40cm), it has plus-shaped construction that seems to be raised 50cm above the ground. The enclosed arms have walls that provide the animals with a sense of protection, whereas the open arms are devoid of both walls and protective boundaries. A centre square of 10×10cm functions as the connection between the arms. The experiment took place during the dark phase of the light cycle, from 9 to 14hours. Each rat plus maze device, facing the open arms, 45minutes after the extracts, control, and standard were supplied orally using an oral canula. The number of times the rats entered and exit open and closed arms over the course of five minutes, as well as the amount of time they spent in each arm type, were recorded. To eliminate any potential bias brought on by the previous animal’s odour, the surface was cleaned with 5% alcohol each time an animal was put. Every precaution was taken to ensure that the animals wouldn’t experience anxiety as a result of external stimulation^{8,9,10,11,12,19,20}.

Model for anxiety light dark box:

The apparatus was made up of two 20×10×14cm transparent black plastic boxes. There was an open door between the two boxes, so the rats could go between them. A 100W light bulb, positioned 30cm from the floor of the glass box, served as the room’s light source. The hole-facing lit box contained a rat. Shortly after the mouse entered the dark box, a 5-minute video was captured showing the movements of the light box and the rodent’s stay within. Every trial was followed by a thorough cleaning of the apparatus. The total number of occurrences within each box as well its duration level will be recorded^9,4,21,22.

Statistical Analysis:

Results were expressed as Mean±SD the differences between experimental groups were compared using one-way Analysis of variance (ANOVA) in GraphPad Prism version 10. Results were statistically significant when P<0.05.¹³

RESULT:

Acute Toxicity Study:

There was neither aberrant behaviour nor clinical signs were displayed by the Wistar rats.

Psychochemical’s Screening of Plant Product:

The yield of aqueous and ethanolic extract was showed in Table 2 whereas Table 3 provides a summary of the photochemical contents found in the aq. extract of Rubia cordifolia. The photochemical present in the extract include phenolics, alkaloids, tannins, flavonoids¹⁴.

Table 2: Extractive yield of various extract of Rubia cordifolia leaf

S. N	Extractive Yield	%w/w
1.	Water soluble extractive value	12%
2.	Alcohol soluble extractive value	10.4%

Table 3: Phytoconstituent of leaf of Aq. Extract of Rubia cordifolia

S.N.	Test Performed	Aqueous Extract
1.	Alkaloids	++
2.	Saponin	+++
3.	Flavonoids	++
4.	Glycosides	+
5.	Terpenoids	+
6.	Tannin and phenolic compounds	++

++: moderately present, +: slightly present

Table 4 and Figure 2 reported that the administration of Diazepam (2mg/kg) significantly increased the proportion of indices in the open arm and the amount of time in the same arm (P<0.0010) as compared to the distilled water-treated group. The duration in the stretched arm was significantly extended by Rubia cordifolia aqueous extract at 100mg/kg (P<00.01) and 200mg/kg (P<0.05). Additionally, there was a significant increase in incidence in the open arms at 100mg/kg (P<0.050) and 200mg/kg (P<0.010).

Diazepam (2mg/kg) visibly increases the amount of time spent in the lighting compartment compared to the distilled water-treated group (P<0.001) (Table 5). The amount of time spent in the light-filled compartment increased noticeably after administration (P<0.050). The anxiolytic effects of 100 and 200mg/kg of Rubia cordifolia leaf aqueous extract were contrasted with those of a distilled water-treated group and the control groups shown in Table 5 and Figure 3.

Figure 3: Result of Rubia cordifolia leaf extract in elevated plus maze (mean±SD, n=4)

DISCUSSION:

Most of the drugs used in modern medicine are derived from medicinal plants. Other therapy techniques are being examined due to the unfavourable side effects of benzodiazepines, which have been used extensively over the past 50 years to treat a range of anxiety disorders. The utilization of medicinal plants is a helpful tool for finding new ways to treat certain ailments. Although diazepam and flavonoids share molecular similarities, Rubia cordifolia has an anxiolytic effect that may be like that of diazepam, which works by way of gamma-aminobutyric acid (GABA). The anti-anxiety qualities of Rubia cordifolia leaves were assessed using the widely used elevated plus maze and light-dark box models. These models were chosen due to their simplicity, effectiveness, and lack of training time. Additionally, handling the animal does not cause it any distress. Currently, the E.M.P. test is advised for anxiolytic medications. It is a well-researched animal model that functions effectively in mice and rats. The major discovery that forms the basis of the idea is that animals in an open, elevated maze display an approach-avoidance conflict, which shows up as an exploratory-cum-fear drive. Because they are acrophobic, or afraid of heights, the animals feel uneasy on the raised plus-maze. Reduced movement, as indicated by the amount of time the animal spends in the open arms, is the ultimate indication of fear and anxiety in animals. In the E.P.M. and light-dark box test, the effects of Rubia cordifolia aqueous extract at 100 and 200mg/kg were nearly identical to those of diazepam at 2 mg/kg. The animals in the control group appear to be exhibiting all signs of anxiety since they spend the least amount of time on average in the open arms of an E.P.M. A significant increase in entries into the light chamber and a decrease in time and entries into the dark chamber were the outcomes of administering 200 mg/kg of Rubia cordifolia leaf extract. Similar increases in time spent and entry in the light room were observed with diazepam, indicating that the Light-Dark model had anti-anxiety effects. The results of the study showed that, out of all the extracts examined, 200 mg/kg of aqueous extract had the strongest anxiety activity. This dosage was comparable to diazepam since the effects of the extract and the standard drug were statistically equal.

CONCLUSION:

This study shows that the aqueous leaf extract of Rubia cordifolia can help reduce anxiety in rats. In both the Elevated plus Maze and Light-Dark Box tests, rats treated with the extract, especially at the higher dose of 200mg/kg, spent more time in open or brightly lit areas, suggesting they felt less anxious. These calming effects were like those seen with diazepam, a commonly used anti-anxiety drug. The extract contains natural compounds like flavonoids and alkaloids, which may be responsible for these effects. Overall, these results suggest that Rubia cordifolia could be a promising natural option for managing anxiety. More research is needed to understand exactly how it works and whether it could be safe and effective for humans.

ACKNOWLEDGEMENT:

The authors would like to acknowledge the Shambhunath Institute of Pharmacy, Prayagraj U.P., for providing facilities to carry out this research work.

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Received on 18.08.2025 Revised on 04.10.2025

Accepted on 14.11.2025 Published on 31.01.2026

Available online from February 07, 2026

Asian J. Research Chem.2026; 19(1):1-5.

DOI: 10.52711/0974-4150.2026.00001

This work is licensed under a Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License. Creative Commons License.

Group	Treatment	Dose (orally)	Time spent in open arm (sec.)	No. of entries in open arm
1.	Distilled water	10 ml/kg	45±5	3±1
2.	mCPP	0.5 mg/kg	12± 2	1± 0.3
3.	Diazepam	2 mg/kg	120±8**	8±2**
4.	Aq. Extract of Rubia cordifolia	100 mg/kg	85±6*	5±1*
5.	Aq. Extract of Rubia cordifolia	200 mg/kg	105±7**	7±2**

Group	Treatments	Dose (orally)	Average time in light box (sec.)	No. of transition
I	Distilled water	10 ml/kg	75.2 ± 5.1	6.2 ± 0.4
II	mCPP	0.5 mg/kg	42.5 ± 4.7	3.1 ± 0.5
III	Diazepam	2 mg/kg	110.4 ±6.9*	9.4 ± 0.6*
VI	Aq. Extract of Rubia cordifolia	100 mg/kg	89.3 ± 5.6	7.5 ± 0.7
V	Aq.Extract of Rubia cordifolia	200 mg/kg	102.6 ± 6.2*	8.5 ± 0.5*

INTRODUCTION:

Table 5: Anti-anxiety activity of rat in light and dark box test